2.instructions For Use - Ezplex SARS-CoV-2 G Kit PDF

Ezplex® SARS-CoV-2 G Kit
Ezplex®
SARS-CoV-2 G Kit
Instructions for Use (IFU)
Rx USE ONLY
100Tests / 200Tests
Manufacturer:
SML GENETREE Co., Ltd.
6F, Hanmaeum Bldg, 225 Baumoe-ro, Seocho-gu, Seoul, 06740
Republic of Korea
TEL : +82-2-2057-7900
FAX : +82-70-7425-3950
Website : http://www.genetree.co.kr
IFU-013(rev.3, June-26-2020) SML GENETREE Co., Ltd. 6F, Hanmaeum Bldg., 225 Baumoe-ro,
Page1 / 20 Seocho-gu, Seoul, Republic of Korea
Ezplex®
SARS-CoV-2 G Kit
For Use with :
Sample Types Extraction Platforms PCR Platform
Upper Respiratory
CFX96
Swabs
Real-time PCR
(Nasopharyngeal/ Instrument
It is recommended that QIAamp DSP
Oropharyngeal) (Bio-Rad)
Virus Spin Kit (Qiagen GmbH) shall be
ABI 7500 Real-time
Lower Respiratory used for RNA extraction.
PCR Instrument
Specimen (Thermofisher
(Sputum) Scientific)
Contents
1. Intended Use ························································································································4
2. Kit Components ·····················································································································4
3. Storage & Transport Conditions ···························································································5
4. Identification of Material & Devices Required but Not Provided ··········································6
5. Background Information ·······································································································6
6. Product Description ···············································································································7
7. Warning and Precautions ······································································································7
8. Limitation of Use ···················································································································8
9. Procedure ······························································································································9
10. Performance Evaluation ···································································································· 16
11. Symbols and Information ·································································································· 17
12. Technical Support ·············································································································· 17
1. Intended Use
The Ezplex® SARS-CoV-2 G kit is a Reverse Transcription Real-time PCR test intended for the qualitative detection of
nucleic acid from SARS-CoV-2 in nasopharyngeal, oropharyngeal and sputum specimens collected from individuals
suspected of COVID-19 by their healthcare provider. The Ezplex® SARS-CoV-2 G kit is intended for use by qualified
clinical laboratory personnel specifically trained and instructed in RT Real-time PCR techniques and in vitro diagnostic
procedures.
2. Kit Components
* 100 test
No. Kit Component Volume Lid Remarks
1 RQ Mixture 1 vial, 1000uL RQ

2 P+P 1 vial, 500uL P+P
3 Positive Control 1 vial, 50uL PC
4 Negative Control 1 vial, 50uL NC
5 Internal Control 1 vial, 20uL IC
* 200 test
No. Kit Component Volume Lid Remarks
1 RQ Mixture 2 vials, 1000uL RQ

2 P+P 2 vials, 500uL P+P
3 Positive Control 2 vials, 50uL PC
4 Negative Control 2 vials, 50uL NC
5 Internal Control 2 vials, 20uL IC
3. Storage & Transport Conditions

3.1 Storage
storage
No. Reagent Name Status Shelf life
Condition
1 RQ Mixture Opened/Unopened -20
2 P+P Opened/Unopened -20
6 months from date of

3 Positive Control Opened/Unopened -20
manufacture
4 Negative Control Opened/Unopened -20
5 Internal Control Opened/Unopened -20
3.2 Storage and transport conditions
① Products that are packaged shall be stored in a storage freezer.
② To transport the products to a client, the products shall be put in a cooler with dry ice so that the
products are not exposed to the ambient temperature.
When sending the products via a courier service, product boxes shall be wrapped with bubble wraps
③ before
putting in a cooler, ices packs shall be stacked on the products, and then dry ice shall be filled to cover
more than 1/3 of the entire box so that temperature change during delivery is minimized.\
3.3 Efficacy of Freezing and Thawing

It is a principle to use this only once, but when it is repeated thawing and freezing, should not exceed
5 freeze-thaw cycles.
4. Identification of Material & Devices Required but Not Provided

No. Name CAT No. Remarks
1 Any applicable 96 well PCR plate plastics for MLL9651 Bio-Rad
CFX96 system
2 Any applicable PCR plate sealing film for MSB1001 Bio-Rad
CFX96 system
3 MicroAmp Optical Adhesive Film 4311971 Thermofisher
Scientific
4 MicroAmp Optical 96-well Reaction Plate N8010560 Thermofisher
Scientific
5 MicroAmp Optical 8-tube strip(0.2mL) 431567 Thermofisher
Scientific
6 MicroAmp Optical 8-cap Strip 4323032 Thermofisher
Scientific
7 Qiagen QIAamp® DSP Viral RNA Mini Kit 61904 Qiagen
5. Background Information
The target is important to be screened for patient displaying relevant symptoms of COVID-19.
Reliable and regular diagnosis of individuals for this target will help to ensure a reduction in the
spread of infections but also help rapidly treat infected patients.
COVID-19 is a contagious, novel strain of Coronavirus that emerged from Wuhan, China in December
2019. The virus is thought to be zoonotic origin and likely to have spread from large seafood and animal
markets by human-animal contact in the city of Wuhan. The virus causes respiratory infection, with
symptoms including fatigue, fever, shortness of breath, respiratory failure, renal failure and death. As of
the time of writing, February 2020, globally confirmed cases exceed 60,000.
Patients can become infected with COVID-19 by person-person contact (through contact with a
contaminated environment or person). The young and old (and other immunocompromised individuals)
are most at risk of serious complications however, approximately 20% of those infected can develop a
critical condition.
6. Product Description
The Ezplex® SARS-CoV-2 G Kit is a real-time reverse transcription polymerase chainreaction (rRT-PCR) test.
The 2019-nCoV primer and probe set(s) is designed to detect RNA from the SARS-CoV-2 Virus in Upper
Respiratory(Nasopharyngeal/Oropharyngeal) swabs and Lower Respiratory specimens(Sputum) from patients with
signs and symptoms of infection who are suspected of COVID-19.
6.1 Negative control

The negative control is needed to serve as a general control for exogenous nucleic acid contamination and is used
tomonitor of cross-contamination during RNA extraction and PCR reaction setup. It shall be used at least once in
single test.
6.2 Positive control

The positive control is needed to serve as a general control for monitoring whether PCR is performing correctly and
is used to test for the presence of inhibitors in the sample of the efficiency of the polymerase chain reaction itself
using the primer set that detects template DNA sequences. It shall be used atleast once in single test together with
NTC and it includes synthesized plasmid DNA of each subject gene targets.
6.3 Internal control
Synthesized plasmid DNA of Homo sapiens BCR activator of RhoGEF and GTPase (BCR) region is needed to
provide assurance that clinical specimens are successfully amplified and detected and is used in the way of adding
reaction master mixture during PCR preparation procedure.
7. Warning and Precautions
① This product is intended for in-vitro diagnostic use only, and shall be used by clinical expert such as clinical
pathologist and medical technologist.
② Insufficient test results, such as inconclusive result, through this product shall be confirmed together with additional
diagnostic measures,
③ All product components shall be taken out just before use and shall be stored in a freezer(below 20 )
immediately
after use.They are exposed as little as possible to the ambient temperature.
④ Beware of carry-over contamination since the Real-time PCR has a high sensitivity.
⑤ Repeated freezing and thawing of reagent and specimen shall be avoided because they may affect the test
sensitivity.
⑥ Beware of microbe contamination when dividing the reagent and it is recommended to use a sterilized disposable
filter tip.
⑦ Beware not to touch the reagent container cap or the inner side of PCR tube cap with your hands.
⑧ It is prohibited to mix the products from different Lots even in case of the same product s reagent.
⑨ Do not use the product if the use authorization is expired.
⑩ Tests shall be performed in accordance with the Guideline for Laboratory Biosafety and the Laboratory Safe
Management Manual.
⑪ While handling the specimen, beware of infection through skin or inhalation. In case of human exposure, the part
shall be immediately cleansed with running tab water and medical attention shall be sought immediately for
symptoms including high fever and rashes.
⑫ Tests shall be performed in accordance with the Guideline for Laboratory Biosafety and the Laboratory Safe
Management Manual, and all spaces shall be thoroughly sterilized using 70% Ethanol or 0.5% sodium
hypochlorite.
8. Limitation of Use
① The Ezplex® SARS-CoV-2 G Kit has been validated for use with Upper Respiratory(Nasopharyngeal/Oropharyngeal)
swabs and Lower Respiratory specimens(Sputum)samples run on the Bio-Rad CFX96 Real-Time PCR and ABI 7500
Real-time PCR Instrument.
② The procedures in this handbook must be followed, as described. Any deviations may result in assay failure or cause
erroneous results.
③ Good laboratory practice is required to ensure the performance of the kit, with care required to prevent contamination
of the kit components. Components should be monitored for contamination and any components thought to have
become contaminated should be discarded as standard laboratory waste in a sealed pouch or zip-lock plastic bag.
④ All samples should be handled as if they are infectious following proper biosafety precautions.
⑤ Interpretation of results must account for the possibility of false negative and false positive results.
⑥ False negative results may be caused by:

- Unsuitable collection, handling and/or storage of samples.
- Sample outside of viraemic phase.
- Failure to follow procedures in this handbook.
- Use of unauthorized extraction kit or PCR platform.
⑦ False positive results may be caused by:

- Unsuitable handling of samples containing high concentration of COVID-19 viral RNAor positive control template.
- Unsuitable handling of amplified product.
⑧ All results should be interpreted by a health care professional in the context of patient medical history and clinical
symptoms.
⑨ This test cannot rule out diseases caused by other pathogens.
⑩ A negative result for any PCR test does not conclusively rule out the possibility of infection.
9. Procedure
9.1 Preparation
9.1.1 Specimen Preparation and Storage
① UpperRespiratory(Nasopharyngeal/Oropharyngeal) swabs and Lower

Respiratoryspecimens(Sputum)samples shall be used for the test.
② It is recommended that swab specimens shall be used immediately after collection. However, the specimens
can be stored maximum 3 days at 2-8 in a fridge or maximum 2 months at 20 in a freezer if immediate
use is not achievable.
③ Specimens shall be divided into amounts required for one testing and stored at 20 in a freezer so as to
avoid from thawing repeatedly.
④ Specimens that are no longer needed shall be put in a container for liquids and disposed as liquid medical
waste.
⑤ Specimen collection
- Specimens shall be collected in a dedicated container which shall be sealed to prevent leakage.
- Adequate protective gears such as gloves and gowns shall be used to handle the specimens.
- Protective glasses, masks, or aprons shall be worn if protection is required against specimen splatter.
9.1.2 Pre-test Preparations
① Reagents shall be stored at 20 and shall avoid repeated freezing and thawing.
② Reagents shall be used after completely thawed.
③ Since RNA can be degraded from the positive control, it is recommended that the reagents shall be divided into
amounts required for 1-2 tests and stored in a freezer.
④ Equipment required for testing:

CFX96 Touch Real-time PCR Detection system (Bio-Rad)
Applied Biosystems 7500 Real-time PCR system (Thermofisher scientific)
9.2 Test Procedure
9.2.1 Specimen Pretreatment
While it is possible to use various ways and kits adopted in laboratories to extract RNA and apply on this product,
it is recommended that QIAamp DSP Virus Spin Kit (Qiagen GmbH) shall be used for RNA extraction and users
shallfollow the protocol included in the Kit Handbook. After being extracted, RNA shall be stored at 20 ± 2 in
a freezerand shall be divided into amounts required for 1-2 tests since RNA can be degraded.
9.2.2 Real-time PCR Amplification
9.2.2.1 Making reagent master mix solution
①Refer to the tables below and make PCR master mix solution according to the number of samples to be tested
(See Table 1, 2).
Table 1. In case a separate IC is included in specimen extraction(unit: uL)
Component Capacity
RQ Mixture 10
P+P 5
Total 15
Table 2. In case a separate IC is not included in specimen extration(unit: uL)
Component Capacity
RQ Mixture 10
P+P 5
IC 0.1
Total 15.1
② Divide 15uL of PCR master mix solution in PCR tubes, add 5uL of the RNA specimen in each tube, and mix them
well.
③ Both positive control and negative control shall be tested for accuracy.
9.3 Set up the device with below conditions
Step Temparature / Time Cycle
25 / 2 min
Hold 50 / 30 min 1 Cycle
95 / 5 min
95 / 15 sec
Cycle 40 Cycles
60 / 45 sec
9.4 Result
9.4.1 Fluorescent thresholds for detection targets shall be set as the table below.
Device FAM Cy5 VIC(HEX)
CFX96 500 250 500
AB7500 0.4 0.1 0.05
9.4.2 Result Interpretation shall be refer to the table below.
FAM (RdRp) CY5 (N) VIC/HEX(IC) Result* Remark
<40 <40 Any Positive
<40 40 Neg Any Inconclusive
40 Neg <40 Any Inconclusive
40 Neg 40 Neg <38 Negative
Retest
40 Neg 40 Neg 38 Neg Invalid
after re-extraction
* The result is judged as Positive only when it is detected all of RdRp, and N gene. Further confirmatory test shall be
necessary if the result is judged as "Inconclusive".
9.5.3Positive & Negative control range

Due to PCR instruments managing in all different conditions, the individual fluorescence thresholds are changeable if
controls are deviated from measured ranges as below table.
<CFX96 Version>
Control RdRp Ct(FAM) N Ct(Cy5) IC Ct(VIC,HEX)
PC 24.0 ~ 27.0 22.0 ~ 25.0 Any
NC Neg Neg 28.0 ~ 32.0
<7500 Version>
Control RdRp Ct(FAM) N Ct(Cy5) IC Ct(VIC,HEX)
PC 26.0 ~ 29.0 23.0 ~ 26.0 Any
NC Neg Neg 26.0 ~ 30.0
9.6 Software analysis(Genetree Viewer)
9.6.1 Software installation
① Before installing analysis software, the Microsoft Visual C++ 2015 Redistributable(x86)' shall be installed in
advance.
② After pre-installation step, click on 'Run as administrator' of file 'Setup.exe' in the installationfolder of 'Genetree
Viewer'.
NOTE : If the installation is not possible due to the antivirus software, the installation can be performed by temporarily stopping or not
running the antivirus software and proceeding with the installation.
NOTE : Please contact 'genetree@genetree.co.kr' to acquire 'Genetree viewer' software.
③ If the installation is completed, the run file of analysis software can be found in the 'Start menu' as below.
9.6.2 Software Analysis (CFX96 Real-time PCR Instrument)
① Check that PCR is finished and click to enable apply fluorescence drift correction in the CFX96 manager
software.
Note : This configuring step is only needed to CFX manager software 3.1 version. In case of use CFX manager 1.6, the step '①' and '②'
can be skipped.
② Readjust the threshold for the positive control and negative control and click Save in the CFX96 manager software.
③ Click 'Export All Data Sheets' from CFX manager software s Tool menu to convert the test data into an excel
spreadsheet (Create a folder and save the file in it).

Note: Select Excel 2003 or Excel 2007 according to the Excel specifications.
<CFX manager software: 3.1 version>
<CFX manager software: 1.6 version>
④ Run the anal sis software (Genetree Viewer), press Open on the upper left to navigate the folder where the
converted excel file is saved, and open the file with name that ends with 'Quantitation Amplification Results.'
⑤Click Please select a kit menu at the top of the screen and select an appropriate item for the tested panel
(2019-nCoV real-time PCR: CFX1.6 and 3.1).
9.6.3 Software Analysis (ABI 7500 Real-time PCR Instrument)
①Run the 7500 software, click the 'Export' in the export menu.
② When the Export Properties window appears, select the 'Result' and 'Amplification' data in the '1. Select data to
export' checkbox, specify the Excel file name in 'ExportFile Name', and proceed the export of data files.
③ Run the 'Genetree Viewer' software, and load the exported data files via 'Open' menu.
④ When it successfully loads the files, click the 'Please select a kit' and select the appropriate test item as below.
⑤ As it is shown in the below image, results for each well can be checked according to the selected Kit component.
Note: Refer to the below table for description of the results.
No. Description
① Positive/Negative results by well are indicated in '+', '-' respectively.
② Ct and fluorescent values of the results for each well are plotted on a graph.
Ct values of the results for each well are indicated numerically and qualitative results
③ are printed.
④ Analysis results are converted into an excel spreadsheet.
10. Performance Evaluation

10.1 Analytical Sensitivity(Limit of Detection)
Using standard SARS-CoV-2 material(Vircell, MBC137-R), it was serially spiked in both of Upper
respiratory(Nasopharyngeal) and Lower respiratory specimens(Sputum), and RNA were extracted from those
prepared specimens by QIAamp DSP Virus Spin Kit(Qiagen). The test was performed 20 times on every diluted
concentrations and the limit of detection is estimated as the copy number which is equal to or greather than 95% of
samples.
CFX96 ABI7500
Target
Nasopharyngeal Sputum Nasopharyngeal Sputum
RdRp 1.25 copies/uL 1.25 copies/uL 1.25 copies/uL 0.625 copies/uL
N 1.25 copies/uL 0.625 copies/uL 1.25 copies/uL 1.25 copies/uL
10.2 Analytical Sensitivity(Inclusivity)

In silico analysis for all sequences of SARS-CoV-2 Virus, available from NCBI, was conducted by comparing the
primers and probes of the Ezplex® SARS-CoV-2 G Kit. In the in silico anaylsis through NCBI sequences, generated
data as shown in table below.
Target Gene Primer/Probe Percent Identities
Forward Primer 100 %
RdRp Reverse Primer 100 %
Probe 100 %
SARS-CoV-2 Virus
Forward Primer 100 %
N Reverse Primer 100 %
Probe 100 %
10.2 Analytical Specificity
10.2.1 Cross reactivity

The 24 species of microorganisms, which are expected of cross reactivity, are chosen for the test and the test
repeated three times on every chosen species. As a result of the test, there were no cross reactivity as those were
observed as all negative result.
Microorganisms Cocentrations Microorganisms Cocentrations
Adenovirus 1X104 copies/uL Legionella pneumophila 1X104 copies/uL
Human metapneumovirus 1X106 copies/uL Mycobacterium tuberculosis 1X104 copies/uL
Human parainfluenza virus 1 1X104 copies/uL Streptococcus pneumoniae 1X104 copies/uL
Human parainfluenza virus 2 1X104 copies/uL Bordetella pertussis 1X104 copies/uL
Human parainfluenza virus 3 1X104 copies/uL Mycoplasma pneumoniae 1X104 copies/uL
Human parainfluenza virus 4 1X104 copies/uL Candida albicans 1X104 copies/uL
Influenza A virus 1X104 copies/uL Staphylococcus aureus 1X104 copies/uL
Enterovirus 71 type 1X104 copies/uL Human coronavirus OC43 1.41X106 copies/uL
Human respiratory syncytial
1X104 copies/uL Human coronavirus NL63 1X106 copies/uL
virus A
Human respiratory syncytial Severe acute respiratory
1X104 copies/uL 1X106 copies/uL
virus B syndrome
Human rhinovirus 1X104 copies/uL Middle East respiratory 1X106 copies/uL
syndrome
Chlamydophila pneumoniae 1X104 copies/uL
Haemophilus influenzae 1X104 copies/uL
10.2.2 Interference
The interference materials were prepared with 3 materials of endogenous - Albumin (0.24g/mL), Hemoglobin
(0.2g/mL), Bilirubin (0.05mg/mL) and 3 materials of exogenous -Mupirocin(20mg/mL), Tobramycin (80mg/mL),
Zanamivir (250ug/mL) those were tested three times with and without positive materials of SARS-CoV-2 diluted in low
concentration. As a result of the test, there were no interference by observing the coefficient of variation(CV) value
which were less than 5% in all cases.
10.3Clinical Evaluation
In the clinical evaluation study, selected left-over archived samples from symptomatic patients suspected of COVID-19
infection. Specimens were previously subjected for SARS-CoV-2 testing and then stored at a clinical laboratory in
South Korea prior to including in this study. A total of 60 samples (30 upper respiratory
samples(nasopharyngeal/oropharyn-geal), 30 lower respiratory samples); 30 positive samples (15 upper respiratory
samples, 15 lower respiratory samples) and 30 negative samples(15 upper respiratory samples, 15 lower repiratory
samples) were tested. The purpose of this clinical evaluation was to assedd the clinical performance of Ezplex ®
SARS-CoV-2 G Kit.
For this study, extraction was performed using the QIAamp DSP Virus Spin Kit(Qiagen). Real-time RT-PCR was
perofrmed using the CFX96 Real-time PCR Instrument(Bio-Rad).
All specimens were evaluated with the Ezplex® SARS-CoV-2 G Kit.and the comparator real-time PCR assay. The
coparator assay was Allpex 2019-nCoV Assay(Seegene) which was authorized by FDA EUA in April 21, 2020.
The results from testing of individual speicmens are shown as below.
Control Group Control Group

Upper Respiratory Lower Respiratory
Positive Negative Total Positive Negative Total
Experiment Positive 15 0 15 Experiment Positive 15 0 15
group Negative 0 15 15 group Negative 0 15 15
Total 15 15 30 Total 15 15 30
Positive agreement Positive agreement
100 % (78.2 ~ 100 %) 100 % (78.2 ~ 100 %)
(95% CI) (95% CI)
Negative agreement Negative agreement
100 % (78.2 ~ 100 %) 100 % (78.2 ~ 100 %)
(95% CI) (95% CI)
11. Symbols and Information
Symbol Meaning Symbol Meaning
In-Vitro Diagnostic
Storage Temperature
Medical Devices
Expiration date Product User Manual
Catalogue Number Manufacturer
Keep away
Lot Number from sunlight
(P+P)
Contents sufficient for
<n> tests
12. Technical Support

For Technical support, please contact our dedicated technical support team on:
Email : genetree@genetree.co.kr
Tel: +82-2-2057-7900
Fax: +82-70-7425-3950

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2.instructions For Use - Ezplex SARS-CoV-2 G Kit PDF

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Ezplex® SARS-CoV-2 G Kit

Instructions for Use (IFU)

For Use with :

Sample Types Extraction Platforms PCR Platform

2. Kit Components ·····················································································································4

3. Storage & Transport Conditions ···························································································5

4. Identification of Material & Devices Required but Not Provided ··········································6

5. Background Information ·······································································································6

6. Product Description ···············································································································7

7. Warning and Precautions ······································································································7

8. Limitation of Use ···················································································································8

10. Performance Evaluation ···································································································· 16

11. Symbols and Information ·································································································· 17

12. Technical Support ·············································································································· 17

No. Kit Component Volume Lid Remarks

1 RQ Mixture 1 vial, 1000uL RQ

No. Kit Component Volume Lid Remarks

1 RQ Mixture 2 vials, 1000uL RQ

3. Storage & Transport Conditions

1 RQ Mixture Opened/Unopened -20

2 P+P Opened/Unopened -20

6 months from date of

4 Negative Control Opened/Unopened -20

5 Internal Control Opened/Unopened -20

3.2 Storage and transport conditions

① Products that are packaged shall be stored in a storage freezer.

3.3 Efficacy of Freezing and Thawing

4. Identification of Material & Devices Required but Not Provided

6.1 Negative control

6.2 Positive control

6.3 Internal control

7. Warning and Precautions

⑨ Do not use the product if the use authorization is expired.

⑥ False negative results may be caused by:

⑦ False positive results may be caused by:

⑨ This test cannot rule out diseases caused by other pathogens.

9.1.1 Specimen Preparation and Storage

① UpperRespiratory(Nasopharyngeal/Oropharyngeal) swabs and Lower

9.1.2 Pre-test Preparations

② Reagents shall be used after completely thawed.

④ Equipment required for testing:

9.2 Test Procedure

9.2.1 Specimen Pretreatment

9.2.2 Real-time PCR Amplification

9.2.2.1 Making reagent master mix solution

(See Table 1, 2).

Table 1. In case a separate IC is included in specimen extraction(unit: uL)

Table 2. In case a separate IC is not included in specimen extration(unit: uL)

9.3 Set up the device with below conditions

Step Temparature / Time Cycle

Hold 50 / 30 min 1 Cycle

Device FAM Cy5 VIC(HEX)

CFX96 500 250 500

AB7500 0.4 0.1 0.05

9.4.2 Result Interpretation shall be refer to the table below.

FAM (RdRp) CY5 (N) VIC/HEX(IC) Result* Remark

<40 <40 Any Positive

<40 40 Neg Any Inconclusive

40 Neg <40 Any Inconclusive

40 Neg 40 Neg <38 Negative

9.5.3Positive & Negative control range

9.6 Software analysis(Genetree Viewer)

9.6.1 Software installation